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Platelets play an important role in hemostasis, inflammation, tumor metastasis, wound healing and defense response, but its routine storage requires a specific temperature and the storage time is generally limited to 5 ~7 d, at the same time, platelet storage damage and bacterial breeding will limit its storage time. In addition, routine transfusion can cause serious adverse reactions such as non-hemolytic fever, allergies, circulatory overload, and transfusion-related acute lung injury. These limit the clinical application of platelet products. However, additives associated with platelet preservation can reduce the likelihood of their occurrence. Therefore, this article reviews the research status of additives related to platelet preservation in recent years, as well as improving platelet storage damage and improving platelet preservation characteristics.
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ObjectiveTo compare the differences in resistance and structure of skin between acupoints and non-acupoints， and to study the difference in skin permeability characteristics of Corydalis Rhizoma total alkaloid patches （CTTP） after administration at Shenque acupoint and non-acupoint， so as to provide experimental support for its clinical acupoint application to prevent and treat chronic pain. MethodTaking corydaline （CD）， tetrahydropalmatine （THP） and corydalis L （CDL） as evaluation indexes， and the quantitative analysis was carried out by high performance liquid chromatography （HPLC）. The mobile phase was methanol-0.04 mol·L-1 phosphoric acid aqueous solution （70∶30， pH 6.0 adjusted with triethylamine）， the detection wavelength was 281 nm. In vitro transdermal test in Franz diffusion cell and in vivo transdermal test were used to study the skin permeability characteristics of CTTP through Shenque acupoint and non-acupoint administration. At the same time， the skin resistance between Shenque acupoint and non-acupoint was measured before and after the administration， and the distribution of the drug in each layer of the skin was compared by freezing sectioning， and visual verification was performed with fluorescence inverted microscope. ResultAfter 24 h of administration， the results of in vivo and in vitro experiments showed that the cumulative permeation and retention of CD， THP and CDL at Shenque acupoint skin were higher than those at non-acupoint skin （P<0.05， P<0.01）， the skin resistance of Shenque acupoint was lower than that of non-acupoint at all time points. The fluorescence microscopic observation results showed that the drug content of each layer of the skin was all Shenque acupoint>non-acupoint， indicating that the skin of Shenque acupoint had better effect on drug penetration and storage than non-acupoint. ConclusionThe 24 h cumulative permeation and retention of CTTP in Shenque acupoint skin are higher than those in non-acupoint skin， and the mechanism may be related to the thin skin， low electrical resistance and large number of hair follicle bodies at Shenque acupoint.
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ObjectiveTo compare the differences in resistance and structure of skin between acupoints and non-acupoints， and to study the difference in skin permeability characteristics of Corydalis Rhizoma total alkaloid patches （CTTP） after administration at Shenque acupoint and non-acupoint， so as to provide experimental support for its clinical acupoint application to prevent and treat chronic pain. MethodTaking corydaline （CD）， tetrahydropalmatine （THP） and corydalis L （CDL） as evaluation indexes， and the quantitative analysis was carried out by high performance liquid chromatography （HPLC）. The mobile phase was methanol-0.04 mol·L-1 phosphoric acid aqueous solution （70∶30， pH 6.0 adjusted with triethylamine）， the detection wavelength was 281 nm. In vitro transdermal test in Franz diffusion cell and in vivo transdermal test were used to study the skin permeability characteristics of CTTP through Shenque acupoint and non-acupoint administration. At the same time， the skin resistance between Shenque acupoint and non-acupoint was measured before and after the administration， and the distribution of the drug in each layer of the skin was compared by freezing sectioning， and visual verification was performed with fluorescence inverted microscope. ResultAfter 24 h of administration， the results of in vivo and in vitro experiments showed that the cumulative permeation and retention of CD， THP and CDL at Shenque acupoint skin were higher than those at non-acupoint skin （P<0.05， P<0.01）， the skin resistance of Shenque acupoint was lower than that of non-acupoint at all time points. The fluorescence microscopic observation results showed that the drug content of each layer of the skin was all Shenque acupoint>non-acupoint， indicating that the skin of Shenque acupoint had better effect on drug penetration and storage than non-acupoint. ConclusionThe 24 h cumulative permeation and retention of CTTP in Shenque acupoint skin are higher than those in non-acupoint skin， and the mechanism may be related to the thin skin， low electrical resistance and large number of hair follicle bodies at Shenque acupoint.
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Early screening is an important means to reduce breast cancer mortality. In order to solve the problem of low breast cancer screening rates caused by limited medical resources in remote and impoverished areas, this paper designs a breast cancer screening system aided with portable ultrasound Clarius. The system automatically segments the tumor area of the B-ultrasound image on the mobile terminal and uses the ultrasound radio frequency data on the cloud server to automatically classify the benign and malignant tumors. Experimental results in this study show that the accuracy of breast tumor segmentation reaches 98%, and the accuracy of benign and malignant classification reaches 82%, and the system is accurate and reliable. The system is easy to set up and operate, which is convenient for patients in remote and poor areas to carry out early breast cancer screening. It is beneficial to objectively diagnose disease, and it is the first time for the domestic breast cancer auxiliary screening system on the mobile terminal.
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Female , Humans , Breast/pathology , Breast Neoplasms/pathology , Diagnosis, Computer-Assisted , Early Detection of Cancer , Ultrasonography , Ultrasonography, Mammary/methodsABSTRACT
Objective To investigate whether silencing UCP2 can sensitize cervical cancer cell line Siha to radiation.Methods Siha cells were transfected with UCP2 siRNA and then irradiated by Xray.The radiosensitivity of Siha cells was verified by colony formation,CCK-8,apoptosis and immunofluorescence assays.The mitochondrial membrane potential and the production of reactive oxygen species (ROS) were detected to further explore the related mechanism.Results RT-PCR and Western blot assays showed that the expression of UCP2 in Siha cells was increased after irradiation and the UCP2 siRNA successfully silenced the expression of UCP in cells.According to the survival curves,the D0,Dq,N and SF2 were 1.54,1.31,2.31 Gy and 0.52 for siUCP2 group,2.50,3.64,4.30 Gy and 0.83 for blank control group,and 3.34,2.16,1.91 Gy and 0.69,for siNC group,respectively.The radiosensitivity enhancement ratio of silent group was 0.62 and 0.46,compared with blank control group and negative control group,respectively.The proliferative activity of cells in the silent group was lower than that in the control group (t =13.2,P＜0.05).Apoptosis levels in the silent group were significantly higher than those in the control group after irradiation (t=3.14,P＜0.05).At 4 h after irradiation,the ROS production in the silent group was significantly higher than that in the control group (t=19.10,P＜0.05).At 24 h after irradiation,the mitochondrial membrane potential of Siha cells in the silent group was significantly lower than that in the control group (t =4.18,P ＜ 0.05) Conclusions The radiosensitivity of Siha cells is enhanced after UCP2 silencing,and thus UCP2 may applicable as a new target for radiosensitization of cervical cancer cells.
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Objective@#To investigate whether silencing UCP2 can sensitize cervical cancer cell line Siha to radiation.@*Methods@#Siha cells were transfected with UCP2 siRNA and then irradiated by X-ray. The radiosensitivity of Siha cells was verified by colony formation, CCK-8, apoptosis and immunofluorescence assays. The mitochondrial membrane potential and the production of reactive oxygen species (ROS) were detected to further explore the related mechanism.@*Results@#RT-PCR and Western blot assays showed that the expression of UCP2 in Siha cells was increased after irradiation and the UCP2 siRNA successfully silenced the expression of UCP in cells. According to the survival curves, the D0, Dq, N and SF2 were 1.54, 1.31, 2.31 Gy and 0.52 for siUCP2 group, 2.50, 3.64, 4.30 Gy and 0.83 for blank control group, and 3.34, 2.16, 1.91 Gy and 0.69, for siNC group, respectively. The radiosensitivity enhancement ratio of silent group was 0.62 and 0.46, compared with blank control group and negative control group, respectively. The proliferative activity of cells in the silent group was lower than that in the control group (t=13.2, P<0.05). Apoptosis levels in the silent group were significantly higher than those in the control group after irradiation(t=3.14, P<0.05). At 4 h after irradiation, the ROS production in the silent group was significantly higher than that in the control group (t=19.10, P<0.05). At 24 h after irradiation, the mitochondrial membrane potential of Siha cells in the silent group was significantly lower than that in the control group (t=4.18, P<0.05).@*Conclusions@#The radiosensitivity of Siha cells is enhanced after UCP2 silencing, and thus UCP2 may applicable as a new target for radiosensitization of cervical cancer cells.
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Objective:To obtain the conditionally replicative adenovirus with triple-targeting Smac overexpression using gene recombination technology,and to explore its effects on the apoptosis and cell cycle progression of MDA-MB-231 cells.Methods:The triple-targeting Smac overexpression vector pShuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55K was constructed by gene recombination technology,which was recombined with the skeleton vector pAdEasy in the BJ5183 bacteria (AdEasy-1+-) to obtain the conditionally replicative adenovirus CRAd.pE-Smac.After the MDA-MB-231 cells were infected with CRAd.pE-Smac,the cancer cells were mimiced into hypoxic status with chemical reagent CoCl2,then control group,CRAd.pE-Smac group,hypoxia group and CRAd.pE-Smac+-hypoxia group were set up;the cells were irradiated with 4 Gy X-rays,and each group was divided into nonirradiation group and irradiation group.The Smac protein expression was detected by Western blotting assay,the apoptotic rates and the percentages of cells at different phases were detected by flow cytometry.Results:The Western blotting results showed that the Smac protein expressions were increased after infection of CRAd.pE-Smac,hypoxia and 4 Gy irradiation,especially in CRAd.pE-Smac +hypoxia+4 Gy irradiation group.The FCM results showed that the apoptotic rates in CARd.pE-Smac,hypoxia,CARd.pE-Smac + hypoxia group were increased compared with control group (P＜0.05 or P＜0.01),and the apoptotic rates of cells irradiated with 4 Gy were significantly increased compared with the unirradiated cells (P＜0.05 or P＜0.01),especially in CRAd.pE-Smac + hypoxia + 4 Gy irradiation group;the percentages of the cells at S and G2/M phases in irradiation groups were significantly increased (P＜ 0.05 or P＜0.01),which had the similar regularity with the apoptotic change.Conclusion:After the MDA-MB-231 cells are infected with the conditionally replicative adenovirus CRAd.pE-Smac and treated with hypoxia and irradiation,the triple-targeting Smac overexpression can be achieved,and it has the role of promoting the cancer cell apoptosis and inducing the G2/M arrest.
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Objective:To detect the changes of the proliferation,cell cycle and apotopsis of liver cancer HepG2cells after treated with Xiaoaiping (XAP) combined with X-rays radiation,and to clarify their anti-tumor mechanisms.Methods:The human liver cancer HepG2 cells at logarithmic phase were selected,and divided into control,XAP,2 Gy radiation and XAP + 2 Gy radiation groups.The HepG2 cells were treated with 75 mg · L-1final concentration of XAP,and they were irradiated with 2 Gy X-rays 12 h later.The cell proliferation activity was detected with CCK8 kits,the cell cycle and apoptotic rates were measured by FCM with PI single staining and Annexin Ⅴ-FITC double staining,and the caspase-3 protein expression was measured by Western blotting method.Results:After the cells were treated with XAP,the proliferation activities in XAP,2 Gy radiation and XAP+2 Gy radiation groups were significantly decreased compared with control group at 12,24 and 48 h (P＜0.05 or P＜0.01);the percentages of cells at S and G2/M phases in XAP,2 Gy radiation and XAP + 2 Gy radiatin groups were significantly increased compared with control group (P＜ 0.05 or P＜ 0.01);the apoptotic rates were significantly increased compared with control group (P＜0.05 or P＜0.01),especially in XAP + 2 Gy radiation group.The Western blotting results showed that caspase-3 was cleaved into 17 000 and 19 000 fragments,and its expression was also increased compared with control group.Conclusion:XAP combined with X-rays radiation could inhibit the proliferation,and induce the G2/M arrest and the apoptosis of liver cancer H epG2 cells;the combination of them shows a synergistic anti-tumor effect.
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Objective:To treat the hepatocellular carcinoma stem cells silenced by Chk-1 with caffeine combined with 4 Gy X-ray irradiaition,and to explore their synergistic killing effects on the hepatocellular carcinoma stem cells by detecting the cell proliferation,cell cycle and apoptosis.Methods:The lentivirus silencing Chk-1 was transfected into the 293T cells;after the HepG2 cells were infected by the lentivirus,the Chk-1 protein expression was detected by Western blotting to confirm the silencing efficency,and non-target control was established,and HepG2-Chk-1 and HepG2-control were named.The cells highly expressed CD133 were cultured by suspension culture method,S-HepG2-Chk-1 and S-HepG2-control were named respectively.After the cells were treated by caffeine,they were irradiated by 4 Gy X-ray;the proliferation activity was measured by MTT,and the cell cycle distribution and the apoptotic rate were measured by PI single staining and AnnexinⅤ-FITC double staining using FCM,respectively.For proliferation,cell cycle and apoptosis assays,there were control,caffeine,4 Gy and caffeine+4 Gy groups.Results:The Western blotting results showed that after the HepG2 cells were infected by lentivirus,the Chk-1 protein expression was significantly decreased,but it was not obvious in non-target control group,it demonstrated that the cell models HepG2-Chk-1 and HepG2-control were obtained successfully.After the HepG2-Chk-1 and HepG2-control cells were cultivated by suspension,the CD133 protein expression were increased,it demonstrated that there were high proportion of CD133+ cells,they were hepatocellular carcinoma stem cells.Compared with control group,the proliferation activities in caffeine group and 4 Gy group were significantly decreased (P＜0.05 or P＜0.01),the percentages of cells at G2/M phage and the apoptotic rates were significantly increased (P＜0.05 or P＜0.01),and the percentage of cells at S phage in caffeine group was significantly increased (P＜0.05);the percentage of cells at G0/G1 phage in 4 Gy group was increased (P＜0.05 or P＜0.01),the effect was more stronger in caffeine+4 Gy group.Compared with S-HepG2-control,the proliferation activities of S-HepG2-Chk-1 ceils in caffeine group and 4 Gy group were decreased,the apoptotic rates were increased (P＜0.05 or P＜0.01),and the percentage of cells at G1/M phage was significantly decreased (P＜0.05 or P＜0.01).Collusion:The hepatoccellular carcinoma stem cells silenced by Chk-1 with positive CD133 are obtained successfully;Caffeine combined with X-ray irradiation can inhibit the cell proliferation and induce the apoptosis,and enhance the G2/M arrest,and both of them have synergistic effects.
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Objective To determine the anatomic distribution of metastatic inguinal nodes in gynecological malignancies,and to explore the delineation of clinical target volume(CTV). Methods A retrospective study was performed among 34 patients with gynecological malignancies and inguinal lymph node metastases. According to the anatomic distribution of metastatic inguinal nodes, CTV covering more than 95% of inguinal lymph nodes and the relationship of inguinal nodes with the femoral vein, greater saphenous vein and its branches, superficial fascia, and deep fascia were analyzed using vascular enhancement images obtained by computed tomography and magnetic resonance imaging as well as 3D reconstruction using the Eclipse Planning System. Results The 34 patients had a total of 145 positive inguinal nodes. In the 131 superficial nodes below the inguinal ligament, 129 were located between the superficial fascia and the deep fascia;the upper group of superficial nodes,containing 25 nodes,was located at 1 cm above the public symphysis and along superficial iliac circumflex vein;the middle group,containing 85 superficial nodes and 11 patients with single superficial node metastasis,was located at the same level of the public symphysis and close to the junction of the saphenous vein and the femoral vein;the lower group, containing 21 superficial nodes,was beneath the public symphysis and along the greater saphenous vein and medial and lateral superficial femoral veins.The 14 deep nodes were located on the medial side of the femoral vein. There were no positive nodes on the posterolateral side of the link between the posterolateral edge of the femoral vein and medial edge of the sartorius muscle. The upper edge of CTV kept 142 lymph nodes beneath the upper edge of the superior pubis ramus and left 3 lymph nodes up to the upper edge of the femoral head. The lower edge of CTV kept 143 lymph nodes above the lower edge of the lesser trochanter and left 2 lymph nodes at 2 cm beneath the lower edge of the lesser trochanter. Conclusions For CTV covering 98% of positive inguinal nodes, the anterior edge is the superficial fascia;the medial edge is composed by the inguinal ligament and the border of medial muscle to the femoral vessels;the posterolateral edge is the link between the posterolateral edge of the femoral vein and the medial edge of the sartorius muscle;the upper edge is the upper border of the femoral head;the lower edge is the lower border of the lesser trochanter.
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Objective:To explore the effect of ionizing radiation on apoptosis of lung cancer H460 cells after ATRX was silenced by RNAi and its mechanism.Methods:The lentivirus expression vectors targeting ATRX were transfected into the 293T cells,and the lung cancer H460 cells were infected with lentivirus twice,and the ATRX silenced cell model was obtained after puromycin positive screening,then they were named as sh-ATRX1-H460,sh-ATRX2-H460,and sh-ATRX3-H460 cells;the sh-control-H460 cells were regarded as control cells.The cells were divided into sh-control-H460 group and sh-ATRX3-H460 group,accroding to the silencing results and were irradiated by 0,2 and 8 Gy X-rays.The expression levels of ATRX,poly(ADP-ribose) polymerase 1(PARP1),and caspase-3 proteins were measured by Western blotting method;the apoptotic rate was measured by flow cytometry and AnnexinⅤ-FITC/PI kits.Results:The lung cancer cell model of sh-ATRX3-H460 silenced by ATRX was obtained successfully.After 2 and 8 Gy X-ray irradiation,compared with before irradiation,the expression level of ATRX protein in sh-control-H460 group was increased,while there was no expression of ATRX protein in sh-control-H460 group;compared with before irradiation,the apoptotic rates of cells in two groups were increased(P<0.05 or P<0.01);the apoptotic rate in sh-ATRX3-H460 group was significantly higher than that in sh-control-H460 group after 8 Gy X-ray irradiation (P<0.01).The expressions of cleaved PARP1 in the cells in both two groups after 2 Gy and 8 Gy X-ray irradiation were increased and showed similar rule.The expression level of procaspase-3 protein in sh-control-H460 group had little change,and it was increased significantly in sh-ATRX3-H460 group after 8 Gy X-ray irradiation.Conclusion:ATRX silencing can be achived by RNAi,then the silencing could increase the apoptosis induced by irradiation and its mechanism may be related to the PARP1-caspase-3 pathway.
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The biologically active constituents in Gynostemma pentaphyllum are dammarane-type glycosides,called gypenosides.They are believed to be the highest contents of this herb,easy to obtain,and mainly active in anti-tumor,controlling the blood glucose,lipid-lowering,cardiovascular protection,etc.The saponins may change into sub-glucoside after hydrolysis,for the intemal acetal glucoside structure is vulnerable to acid,alkali,and enzyme degradation.Through searching the literatures in recent years,this paper summarized the chemical constituents in the hydrolyzed products of gypenosides,for providing references to discover novel and more active lead compounds.
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Objective:To analyze the inhibiting mechanism of microRNA-199a/b-3p(miR-199)on cell proliferation of triple negative breast cancer(TNBC)cells.Methods:Expression of miR199 in BT549 and MDA-MB-231 cells transfected with miR-199a/b-3p was detected by qRT-PCR.The proliferation of BT549 and MDA-MB-231 cells transfected with miR-199 were analysed by MTT as-say.Cell cycle of TNBC cells transfected with miR-199 was detected by Flow Cytometry assay.Results:MiR-199a/b-3p could suppress the proliferation of BT549 and MDA-MB-231 cells.Comparing with normal control,the proliferation rate were up to(41.02±2.34)%and(28.42 ±6.70 )%,and the cell cycle were arrest at G 1 phase.Conclusion: MiR-199a/b-3p could suppress the proliferation of TNBC,and may be a promising anti-cancer drug for TNBC.
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<p><b>OBJECTIVE</b>To investigate the expression of proliferating cell nuclear antigen (Ki-67) in stage III cervical squamous cell carcinoma (SCC) and its correlation with the effect of chemotherapy on sensitivity to radiotherapy.</p><p><b>METHODS</b>In 50 patients with stage III cervical squamous cell carcinoma (SCC), 25 patients were treated with radiotherapy and 25 patients were treated with chemoradiotherapy. The expression of Ki-67 in the biopsy specimens of cervical SCC was detected by immunohistochemistry at diagnosis and after 10 Gy radiotherapy. The correlation of Ki-67 positive cells percentage and chemotherapy with sensitivity to radiotherapy was analyzed.</p><p><b>RESULTS</b>In 25 patients with more than 48% Ki-67 positive cells at diagnosis, the rate of complete response (CR) was 72.0% (18/25). In 25 patients with less than 48% Ki-67 positive cells at diagnosis, the CR rate was 40.0% (10/25), with a significant difference between them (P = 0.023). In 26 patients with more than 31% decrease of Ki-67 positive cells after 10 Gy radiotherapy, the CR rate was 84.6% (22/26). In 24 patients with less than 31% decrease of Ki-67 positive cells after 10 Gy radiotherapy, the CR rate was 25.0% (6/24), showing a significant difference between the two groups (P < 0.001). In the cases of Ki-67<48%, decrease of Ki-67 positive cells of chemoradiotherapy group after 10 Gy radiotherapy was significantly higher than that of the radiotherapy group (P = 0.023). In the cases of Ki-67 ≥ 48%, no difference in the decease of Ki-67 positive cells between the chemoradiotherapy and radiotherapy groups was found (P = 0.173). For the radiotherapy-sensitive patients with CR recently, the 2-year progression free survival (PFS) rate and overall survival (OS) rate were 85.7% and 92.9%, respectively, both were significantly higher than those of radiotherapy-insensitive patients (18.2% and 40.9%, P < 0.05 for both).</p><p><b>CONCLUSIONS</b>In stage III cervical SCC, the expression of Ki-67 before and after treatment with 10 Gy radiotherapy may be used as a biomarker to predict tumor response to radiation, and guide the choice of therapeutic strategies. Yet, the effect of chemotherapy as a radiosensitizer is unconspicuous.</p>
Subject(s)
Female , Humans , Carcinoma, Squamous Cell , Metabolism , Radiotherapy , Chemoradiotherapy , Disease-Free Survival , Epithelial Cells , Immunohistochemistry , Ki-67 Antigen , Metabolism , Neoplasm Staging , Radiotherapy Dosage , Remission Induction , Survival Rate , Uterine Cervical Neoplasms , Metabolism , RadiotherapyABSTRACT
Objective To explore the mechanism of radio-resistance of breast cancer stem cells by investigating the effects of ionzing radiation on the reactive oxygen species (ROS),apoptosis and cycle distribution.Methods The breast cancer MCF-7 cells were suspension cultured in serum-free medium containing a variety of growth factors. There were MCF-7 (breast cancer cells),MCF-7-S (breast cancer stem cells),MCF-7+8 Gy and MCF-7-S+8 Gy groups in the experiment. 4-24 h after 8 Gy irradiation, the ROS levels, percentages of apoptotic cells and percentages of the cells at each cycle phage were measured by FCM with 2′, 7′-dichlorodihydrofluorescin diacetate (DCFH-DA ), Annexin Ⅴ-FITC/PI and PI staining, respectively. Results The breast cancer stem cell microsphere accumulated hundreds of cells were obtained successfully at 7 d after suspension culture with serum-free medium containing a variety of growth factors;the FCM results showed that CD44+CD24- phenotype breast stem cells were up to 75.20%.With the time prolongation,the ROS levels and apoptosis in MCF-7 group and MCF-7-S group showed increasing trendency, and reached for the maximum values at 12 and 24 h;the ROS levels in MCF-7-S group were significantly lower than those in MCF-7 group at 4,8,12 and 24 h (P<0.05 or P<0.01), and the percentage of apoptotic cells in MCF-7-S group was significantly higher than that in MCF-7 group only at 8 h(P<0.05);the ROS levels (4,8,12 and 24 h)and percentage of apoptotic cells(12 h)were significantly increased in MCF-7+8 Gy group (P<0.05),and the percentages of apoptotic cells (4,8,12 and 24 h)in MCF-7-S +8 Gy group were significantly decreased (P<0.05 or P<0.01),but the ROS levels had no obvious change in MCF-7-S+8 Gy.At 12 h,as compared with MCF-7 group,the percentages of the cells at G0/G1 phase and G2/M phase in MCF-7-S group were significantly decreased (P<0.05),and the percentage of the cells at S phase was significantly increased (P<0.05 );the percentage of the cells at G2/M phase in MCF-7+8 Gy group was significantly increased (P<0.05 ), but there were no significant changes in MCF-7-S+ 8 Gy group. Conclusion Ionizing irradiation can cause the increasing of ROS level and apoptosis and G2/M phase arrest in breast cancer cells,but has no obvious effects on the breast cancer stem cells;it indicates that radio-resistance might be related to ROS level,apoptosis and G2/M phase arrest.
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Objective To construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells,and to observe its radiotherapy enhancing effect on tumor cells.Methods The empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time(4,8,12,24 and 48 h)after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5 -5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac mRNA and protein expression levels with RT-PCR and Western blotting methods. The cells were divided into control, pshuttle, pshuttle-Egr-1-hSmac,2.0 Gy irradiation group, pshuttle + 2.0 Gy irradiation and pshuttle-Egr-1-hSmac+2.0 Gy irradiation groups.MTT method was used to evaluate cell proliferation,and the cell survival ability was measured with clone formation assay;Annexin Ⅴ/PI double staining and PI single staining were used to examine the apoptosis and cell cycle of MDA-MB-435 cells. Results There was no Smac mRNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in MDA-MB-435 cells in pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation, and reached the maximum at 24 and 48 h;the Smac mRNA expression levels in MDA-MB-435 cells were increased gradually 24 h after irradiation of 0.5 - 5.0 Gy X-ray with the increasing of irradiation doses, and reached the maximum after 2.0 and 5.0 Gy irradiation. The Smac protein expression levels in pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group. The MTT results showed that the A490 values in 2.0 Gy,pshuttle+2.0 Gy and pshuttle-Egr-1-hSmac groups 24, 48,and 72 h after irradiation were lower than those in control group(P<0.01);the A490 values of MDA-MB-435 cells in pshuttle-Egr-1-hSmac group after 1.0-5.0 Gy X-ray irradiation were lower than those in 0 Gy group (P<0.05 or P<0.01);the survival fraction(SF)in pshuttle-Egr-1-hSmac group was lower than those in control group (P<0.01).The percentages of the cells at G0/G1 and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P<0.01),the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group (P<0.01);the apoptotic rate of the cells in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group (P<0.01).Conclusion X-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the cell survival rate,and induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could significantly increase the radiosensitivity of breast cancer cells.
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Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K carrying early growth response gene-1 (Egr1)promoter and tumor necrosis factor related apoptosis inducing ligand (TRAIL)gene, and to observe the effects of the vector combined with 2 Gy irradiation on the TRAIL expression in MDA-MB-231 cells.Methods Egr-1 promotor sequence was cloned from pMD18 T-Egr1, TRAIL was constructed the downstream of Egr1 promoter, pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K (CRAd.pEgr1-TRAIL)was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231 cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein, respectively. Six groups in the experiment were set up:control, 2 Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p + 2 Gy and CRAd.pEgr1-TRAIL + 2 Gy. Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231 cells transfected with the vector of 5 MOI for 24 h following 2.0 Gy X-rays irradiation began to increase and arrived to the top 8 h later in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231 cells began to increase 6 h after irradiation and reached to the peak 24 h later,then declined 48 h later.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL + 2.0 Gy and other groups at the same time point (P<0.01). Conclusion The recombinant adenovirus vector is obtained successfully, and the TRAIL mRNA and protein expression levels in MDA-MB-231 cells can be increased significantly by the vector combined with 2.0 Gy X-rays irradiation.
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This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellular growth, apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro. The expression of TRAIL and endostatin protein in ECV304 cells was detected by ELISA after the transfection of recombinant plasmid pshuttle-Egr1-shTRAIL-shES and X-ray irradiation. Then MTT assay was used for determining the cellular proliferation, and flow cytometry (FCM) plus Annexin V and propidium iodide (PI) double-staining or PI single-staining were employed for the detection of apoptosis and cell cycle progression. The results showed that expression of TRAIL and endostatin protein exhibited a time- and dose-dependent change in ECV304 cells after pshuttle-Egr1-shTRAIL-shES transfection in conjunction with irradiation. In the TRAIL-endostatin-based single- or double-gene-radiotherapy, the cell viability declined in a time- and dose-dependent manner, the percentage of cells at G(2)/M phase and apoptotic rate was increased, and the percentage of cells at G(0)/G(1) phase was lowered as compared with those receiving radiotherapy alone. Moreover, TRAIL-endostatin-based double-gene-radiotherapy demonstrated better effects on growth inhibition, promotion of apoptosis and induction of cell cycle arrest in ECV304 cells than single-gene-radiotherapy.
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Objective To observe the clinical effects of sufentanil combined with sevoflurane for anesthesia induction in the elderly.Methods A total of 84 patients undergoing elective laparoscopic were randomly divided into 4 groups by dose of Sufentanil (21 cases,each):0.25,0.50,0.75 and 1.00 μg/kg sufentanil.Mean arterial pressure (MAP)and heart rate (HR)were measured at baseline (To),immediately before intubation(T1),immediately,1,3 and 5 min after intubation(T2-5).Intubation scores were also recorded.Results The intubation scores including visual analogue score (VAS) and Ramsay score at 10 min,30 min and 60 min after tube drawing were as the following sequence:1.0 μg/kg ＞ 0.75 μg/kg ＞0.5 μg/kg ＞ 0.25 μg/kg sufentanil (F =5.78.P＜ 0.05).Compared with T0,MAP and HR decreased in each group at T1,while increased at T2 in group A (P＜0.05).As compared with T0,MAP was decreased and HR was slower at T1 in the 4 groups (F=34.99,P＜0.05),but MAP level was increased in 0.25 μg/kg sufentanil group at T2 (F=12.48,P＜0.05).Compared with group of 0.25 μg/kg sufentanil,MAP was reduced in 0.75 μg/kg and 1.00 μg/kg groups at T4 andT5 (F =6.98,6.25,P＜0.05).MAP was also lower in 0.75μg/kg and 1.00μg/kg groups than in 0.50 μg/kg sufentanil group at T2-T5(F=7.08,20.56,P＜0.05).Conclusions Sufentanil of 0.25 0.50 μg/kg combined with sevoflurane can provide excellent intubating conditions and stable hemodynamics during anesthesia induction in patients undergoing gynecologic surgery.
ABSTRACT
Objective: To observe the effects of Tangnaikang (TNK), a compound traditional Chinese herbal medicine, on glucose metabolism and insulin resistance in obese Zucker rats. Methods: Twelve male obese Zucker rats, 6 weeks old, were randomly divided into control group and TNK group (3.24 g/kg) after being fed for 2 weeks. All rats received high-fat diet and 4-week treatment. Body weight and blood glucose were tested every week. Oral glucose tolerance test (OGTT) was performed and fasting insulin level was tested on days 0, 14 and 28. Triglyceride, cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acids (FFA) were tested on day 28. Glucose infusion rate (GIR) was tested by hyperinsulinemic-euglycemic clamp from day 29. The protein expressions of protein kinase B (Akt), phospho-Akt (p-Akt) (Thr308) and glucose transporter protein 4 (GLUT4) in skeletal muscle and GLUT4 in adipose tissue were measured after hyperinsulinemic-euglycemic clamp test. Results: Compared with the control group, the fed blood glucose level and glucose level of OGTT at 120 min had a significant decline in TNK group on day 28, and TNK caused no alteration of the fasting serum insulin, and the GIR increased significantly in hyperinsulinemic-euglycemic clamp study. Furthermore, TNK increased Akt and p-Akt (Thr308) protein expressions in skeletal muscle and decreased the protein expression of GLUT4 in white adipose tissue. Body weight, and triglyceride, cholesterol, LDL-C and FFA contents were slightly decreased in the TNK group, but there were no statistically significant effects. Conclusion: TNK increases the protein expressions of Akt and p-Akt (Thr308) of the signal transduction pathway to influence the translocation of GLUT4 in skeletal muscle and improves glucose metabolism by reducing insulin resistance.